DNA binding by 4-methoxypyrrolic natural products. Preference for intercalation at AT sites by tambjamine E and prodigiosin

Article abstract of DOI:10.1021/jo990944a

The 4-methoxypyrrolic natural products contain a common 4-methoxy-2,2'- bipyrrole chromophore and exhibit promising anticancer, antimicrobial, and immunosuppressive activities. Herein, the ability of two representative members, tambjamine E (1) and prodigiosin (2), to bind calf thymus DNA (CT- DNA), polyd[G-C]₂, and polyd[A-T]₂ has been characterized using absorption and fluorescence spectroscopy. Scatchard plots showed that 1 occupies a site size (n) of ca. three base pairs and possesses affinity constants (K) ranging from 1 to 0.1 x 10⁵ M^-1. Prodigiosin (2) binds DNA by mixed modes, as isobestic points were not evident in titration experiments. The neutral aldehyde precursor 4 was found to possess no measurable DNA binding affinity, indicating that the enamine structure of 1 and the pyrromethene of 2 are essential elements for DNA binding affinity. The enamine of 1 was found to undergo hydrolysis to 4 with a half-life (t( 1/2 )) of 14.5 h at pH 7.4 and 37.5 °C. For the B-ring nitrogen atom of 1, a pK(a) value of 10.06 was also established. From fluorescence spectroscopy it was found that 1, 2, and 4 possess weak emission spectra in water that is increased in nonaqueous solvents. For 1 and 2, DNA binding also increased the emission yield. Energy- transfer measurements suggested an intercalative binding mode, with preference for AT sites. The ability of distamycin to displace 1 and 2 from the helix also suggested that they intercalate from the minor-groove. This specificity differs from other unfused aromatic cations that bind by a minor- groove mode at AT sequences and intercalate at GC sites. Reasons for the specificity displayed by 1 and 2, as well as the implications of our findings to their biological properties are discussed.

Full text of DOI:10.1021/jo990944a

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J . Org. Chem. 1999, 64, 6861-6869
6861
DNA Bin d in g by 4-Meth oxyp yr r olic Na tu r a l P r od u cts. P r efer en ce
for In ter ca la tion a t AT Sites by Ta m bja m in e E a n d P r od igiosin
Matt S. Melvin, David C. Ferguson, Neils Lindquist,† and Richard A. Manderville*
Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486
Received J une 10, 1999
The 4-methoxypyrrolic natural products contain a common 4-methoxy-2,2′-bipyrrole chromophore
and exhibit promising anticancer, antimicrobial, and immunosuppressive activities. Herein, the
ability of two representative members, tambjamine E (1) and prodigiosin (2), to bind calf thymus
DNA (CT-DNA), polyd[G-C]2, and polyd[A-T]2 has been characterized using absorption and
fluorescence spectroscopy. Scatchard plots showed that 1 occupies a site size (n) of ca. three base
pairs and possesses affinity constants (K) ranging from 1 to 0.1 × 105 M-1. Prodigiosin (2) binds
DNA by mixed modes, as isobestic points were not evident in titration experiments. The neutral
aldehyde precursor 4 was found to possess no measurable DNA binding affinity, indicating that
the enamine structure of 1 and the pyrromethene of 2 are essential elements for DNA binding
affinity. The enamine of 1 was found to undergo hydrolysis to 4 with a half-life (t1/2) of 14.5 h at pH
7.4 and 37.5 °C. For the B-ring nitrogen atom of 1, a pKa value of 10.06 was also established. From
fluorescence spectroscopy it was found that 1, 2, and 4 possess weak emission spectra in water
that is increased in nonaqueous solvents. For 1 and 2, DNA binding also increased the emission
yield. Energy-transfer measurements suggested an intercalative binding mode, with preference
for AT sites. The ability of distamycin to displace 1 and 2 from the helix also suggested that they
intercalate from the minor-groove. This specificity differs from other unfused aromatic cations that
bind by a minor-groove mode at AT sequences and intercalate at GC sites. Reasons for the specificity
displayed by 1 and 2, as well as the implications of our findings to their biological properties are
discussed.
In tr od u ction
The tambjamines (i.e., 1),1-4 prodigiosin (2),6-14 and
tetrapyrrole 315 (Figure 1) belong to a family of 4-meth-
oxypyrrolic natural products derived from bacterial and
marine sources that exhibit promising antitumor,5,7,10
antimicrobial,5,10 and immunosuppressive activities.16,17
Representative members shown in Figure 1 are derived
from the aldehyde precursor 4.1,9,10,13,15 Condensation of
* To whom correspondence should be addressed. Tel: (336) 758-
5513. Fax: (336) 758-4656. E-mail: manderra@wfu.edu.
† Institute of Marine Sciences, The University of North Carolina at
Chapel Hill, 3431 Arendell Street, Morehead City, North Carolina.
28557.
(1) Carte´, B.; Faulkner, D. J . J . Org. Chem. 1983, 48, 2314.
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59, 109.
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Antibiot. 1993, 46, 1799.
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F igu r e 1. Structures of 4-methoxypyrrolic natural products
1-3 and aldehyde precursor 4.
Chem. Soc. 1960, 82, 506.
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Soc. 1998, 8305.
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dron Lett. 1968, 641.
4 with the appropriate amine affords the enamine
structure of the tambjamines, i.e., 1.1 Prodigiosin 2 is
colored deeply red due to the planar pyrrolylpyrro-
methene chromophore and is cationic at neutral pH.18
The tetrapyrrole 3 has been identified as the blue
pigment found in tambjamine and prodigiosin frac-
tions.3,15
(16) Nakamura, A.; Nagai, K.; Ando, K.; Tamura, G. J . Antibiot.
1985, 39, 1155.
(17) Tsuji, R. F.; Yamamoto, M.; Nakamura, A.; Kataoka, T.; Magae,
J .; Nagai, K.; J amasaky, M. Antibiotics 1990, 13, 1293.
(18) Hearn, W. R.; Medina-Castro, J .; Elson, M. K. Nature 1968,
220, 170.
10.1021/jo990944a CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/13/1999

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6862 J . Org. Chem., Vol. 64, No. 18, 1999
Melvin et al.
Our interest in the 4-methoxypyrrolic natural products
has been focused on gaining an understanding of their
DNA-targeting properties. Previously, we demonstrated
that tambjamine E (1, Figure 1) facilitates oxidative DNA
cleavage in the presence of copper(II) without the aid of
an external reductant.19 This implied that Cu(I) was
formed reductively through the concomitant oxidation of
1 to a π-radical cation. The tambjamine was found to bind
DNA and Cu(II) effectively, and on the basis of quenching
experiments that argued against participation of a freely
diffusible hydroxyl radical, a copper-oxo species was
proposed to initiate DNA strand scission.19
While our preliminary findings established DNA as a
potential therapeutic target for 1, information is lacking
on the structural basis for DNA binding, specificity of
copper-mediated DNA cleavage (if any), and nature of
oxidative DNA damage (extent of H-abstraction from the
deoxyribose sugars vs oxidation of DNA bases). Deter-
mination of these facets of tambjamine chemistry would
facilitate an understanding of their mode of action in a
biological system. Comparison to the corresponding DNA-
targeting activities of prodigiosin 220 and the tetrapyrrole
3 could then provide the necessary insight for the design
and synthesis of superior polypyrrolic DNA targeting
molecules that may serve as more effective antimicrobial
or anticancer agents.
F igu r e 2. Structures of unfused aromatic cations that bind
DNA.
resent an interesting class of DNA targeting agents. The
bipyrrole coupled to the enamine or pyrromethene enable
these compounds to bind DNA, coordinate redox-active
metal ions such as Cu(II)19 and facilitate oxidative DNA
strand scission in the presence of Cu(II) without the aid
of an external reducing agent.19,20
Here, we focus on the DNA-binding properties of
tambjamine E (1) and prodigiosin (2) as representative
members of the 4-methoxypyrrolic natural products.
These compounds possess unfused aromatic ring systems
that bear cationic charge at physiological pH. This latter
property was found to be critical for DNA binding as the
neutral aldehyde precursor 4 was found to lack the ability
to bind DNA effectively. The fluorescent properties of 1
and 2 permitted us to gain insight into their mode of DNA
binding. These compounds exhibit small emission inten-
sities in water that is dramatically increased in organic
solvents. This phenomenon was also observed for DNA
binding. Energy transfer from the DNA helix (donor) to
the 4-methoxypyrrolic natural product (acceptor) sug-
gested that these compounds bind DNA by intercalation
with a preference for AT sequences. This behavior differs
from other unfused aromatic cations, such as DAPI,21
furamidine,22 and the bithiazole of bleomycin23-26 (Figure
2), whose pharmacological properties have been cor-
related with their DNA targeting activities. For these
compounds, a growing body of evidence implies that they
bind AT sequences by a minor-groove binding mode,
while intercalation is favored at GC sites.21-26
Exp er im en ta l Section
Com p ou n d s. The sample of tambjamine E (1) was obtained
from the organic extracts of the marine ascidian Atapozoa sp.,
as described previously.3,19 Concentrations of 1 were obtained
from UV-vis measurements in methanol: (ꢀ405 ) 24,700 M-1
cm-1).3 Prodigiosin (2) was a gift from the Natural Products
Division of the National Cancer Institute (NCI) and was
received as a dark red powder. Confirmation of prodigiosin
1
structure was obtained by H NMR spectroscopy and electro-
spray mass spectrometry (ES+): [M + H]+ ) 324.2. Stock
solutions of 2 were prepared in dimethyl sulfoxide (DMSO),
and concentrations were determined by UV-vis in 95%
EtOH-HCl (ꢀ535 ) 112 000 M-1 cm-1).18 The bipyrrole aldehyde
precursor 4 was derived from 1 through base hydrolysis using
the protocol described by Faulkner.1 The aldehyde 4 was
obtained as a yellow residue, and its identity was verified by
1H NMR,1,5,10 and ES+: [M + H]+ ) 191.1.1 Concentrations of
4 were obtained from UV-vis (MeOH, ꢀ364
) 6200 M-1 cm-1).1
Bu ffer s. Buffer solutions were prepared from the follow-
ing: MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-
(N-morpholino)ethanesufonic acid, CHES (2-(cyclohexylamino)-
ethanesulfonic acid), CAPS (3-(cyclohexylamino)propanesulfonic
acid (Aldrich), using distilled, deionized water from a Milli-Q
system. Sodium chloride or sodium perchlorate (NaClO4) were
added to adjust the ionic strength, and the pH was adjusted
using NaOH.
Our findings, together with our cleavage results,19,20
show that the 4-methoxypyrrolic natural products rep-
DNA. Calf thymus DNA (CT-DNA, Sigma) was sonicated
and phenol-extracted prior to use, while polyd[G-C]2 and polyd-
[A-T]2 (Sigma) were used without further purification. The
polymer concentrations were determined by applying the
appropriate molar extinction coefficients: ꢀ260 ) 12 824 M-1
cm-1 in base pair (bp) for CT-DNA, ꢀ254 ) 8400 M-1 cm-1 in
base for polyd[G-C]2,27 and ꢀ260 ) 6800 M-1 cm-1 in base for
polyd[A-T]2.28
Kin etic Meth od s. Reactions were followed spectrophoto-
metrically using a Hewlett-Packard (HP-8452) UV-vis spec-
trometer at 392 nm for 1 and 362 nm for 4. Hydrolysis
reactions were initiated by adding 2 µL of a stock solution of
1 (15 mM) in DMSO to a 3-mL cuvette containing 2 mL of
(19) Borah, S.; Melvin, M. S.; Lindquist, N.; Manderville, R. A. J .
Am. Chem. Soc. 1998, 120, 4557.
(20) Prodigiosin 2 also effects oxidative DNA strand scission in the
presence of copper(II) without the aid of an external reducing agent.
Melvin, M. S.; Lindquist, N, Manderville, R. A. Unpublished results.
(21) Wilson, W. D.; Tanious, F.; Barton, H.; J ones, R.; Strekowski,
L.; Boykin, D. J . Am. Chem. Soc. 1989, 111, 5008.
(22) Wilson, W. D.; Tanious, F. A.; Ding, D.; Kumar, A.; Boykin, D.
W.; Colson, P.; Houssier, C.; Bailly, C. J . Am. Chem. Soc. 1998, 120,
10310.
(23) Manderville, R. A.; Ellena, J . F.; Hecht, S. M. J . Am. Chem.
Soc. 1995, 117, 7891.
(24) Wu, W.; Vanderwall, D. E.; Liu, S. M.; Tang, X.-J .; Turner, C.
J .; Kozarich, J . W.; Stubbe, J . J . Am. Chem. Soc. 1996, 118, 10843.
(25) Sucheck, S. J .; Ellena, J . F.; Hecht, S. M. J . Am. Chem. Soc.
1998, 120, 7450.
(27) Wells, R. D.; Larson, J . E.; Grant, R. C.; Shortle, B. E.; Cantor,
C. R. J . Mol. Biol. 1970, 13, 407.
(28) Inman, R. B.; Baldwin, R. L. J . Mol. Biol. 1962, 5, 172.
(26) Cortes, J . C.; Sugiyama, H.; Ikudome, K.; Saito, I.; Wang, A.
H.-J . Biochemistry 1997, 36, 9995.

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DNA Binding by 4-Methoxypyrrolic Natural Products
J . Org. Chem., Vol. 64, No. 18, 1999 6863
buffer (10 mM) equilibrated at 25 or 37 °C. Mixing could be
achieved within 2 or 3 s, and the data, which were analyzed
using the ENZFITTER program, gave good first-order rate
constants for at least 2 half-lives.
P r oton Affin ity. The pH measurements were obtained at
25 °C on a Fisher Scientific Accumet 910 pH meter using
standard glass electrodes. Calibration was done using com-
mercial buffers (BDH, pH 4.00, 7.00, and 10.00, all ( 0.01).
For the pH range 8.5-11.0, the proton affinity of 1 was
determined spectrophotometrically29 at 25 °C using a 1-mL
cuvette containing 1 mL of a 10 mM solution of CAPS, CHES,
or NaOH, with 100 mM NaClO4. To the sample cell was added
2 µL of a 15 mM stock solution of 1 in DMSO. UV-vis spectra
were recorded by the overlay method in the wavelength range
of 250-500 nm.
DNA Bin d in g Affin ity. Apparent equilibrium binding
constants (K) for DNA binding by 1 were determined by UV-
vis at 25 °C using a Hitachi U-2001 UV-vis spectrophotom-
eter. Wavelength scans and absorbance measurements were
made in 3-mL quartz cells of 1-cm path length containing 15
µM 1, 50 mM buffer, and 10 or 100 mM NaCl. The DNA
samples were typically added in 7.5 µM bp aliquots, and
extinction coefficients of both the free and bound drug were
determined at 392 nm, which represented the point of optimal
spectral change.19 For determination of the binding isotherms,
absorbance values were converted into r (moles of 1 bound per
mole of DNA bp) and free ligand concentrations (c). Values
for the fraction of bound drug (R) between 0.2 and 0.8 were
then used to determine K and n (the binding site size in DNA
bp) based on the site exclusion model of McGhee and von
Hippel.30 The ENFITTER program was used to calculate
nonlinear least-squares best-fit values for K and n.
F lu or escen ce Sp ectr oscop y. Fluorescence spectra were
obtained using a Hitachi F-2000 spectrometer at 25 °C. The
following excitation (λexc) and emission (λem) maxima were
utilized: 1, λexc ) 392 nm, λem ) 450 nm; 2, λexc ) 520 nm, λem
) 560 nm; 4, λexc ) 360 nm, λem ) 425 nm. The fluorescence
measurements involving DNA were carried out in 50 mM MES
buffer pH 6.5 containing 10 mM NaCl. Energy transfer from
the polynucleotide to the bound drug was measured from the
excitation spectra of the complex in the wavelength range
F igu r e 3. UV-vis spectroscopic data for the hydrolysis of 1
in 10 mM CHES buffer pH 9.0 containing 100 mM NaClO4 at
25 °C. Arrow indicates loss of tambjamine absorption at 392
nm.
ing Information), which also contains rate constants for
the hydrolysis of 1 at pH 7.4, 37.5 °C, under various ionic
conditions. At 25 °C, rate constants for the hydrolysis
(kobs) increased from 0.857 × 10-5 to 47.7 × 10-5 s-1 as
the pH was varied from 7.4 to 10.1, and at 37.5 °C the
use of NaCl, MgCl2, or NaClO4 had little effect on kobs
.
At the high pH region (9.5-10.5) it was also noted that
1 underwent a change in UV-vis characteristics showing
a blue shift from λmax ) 392 nm to λmax ) 372 nm that
was accompanied by a reduction in peak intensity (Figure
S1, Supporting Information). Prodigiosin 2 also shows a
similar pH-dependent change in absorbance (535 nm in
acidic media, ca. 470 nm in alkali), and this has been
attributed to deprotonation of the pyrromethene chro-
mophore (pKa ) 8.25 in aqueous ethanol, 7.6 in aqueous
dioxane18). The corresponding absorbance change for 1
(Figure S1) permitted determination of a pKa value of
10.06 ( 0.03 (95% confidence interval). This result was
attributed to deprotonation of the B-ring nitrogen atom.5
DNA Bin d in g: Ab sor p t ion Sp ect r oscop y. The
4-methoxypyrrolic natural products have strong absorp-
tion bands in the visible regions that are well suited for
DNA-binding studies. As described previously,19 DNA
binding by 1 leads to a reduction in the extinction
coefficient at 392 nm that is accompanied by a shift to
longer wavelengths. For 1, the changes induced by
binding to CT-DNA, polyd[A-T]2, and polyd[G-C]2 were
similar. Hypochromicity typically fell in the 20-30%
range, and bathochromic shifts were 2-9 nm. Quantita-
tive analysis of the UV-vis data permitted determination
of the intrinsic binding constant (K) and the binding site-
size (n) in DNA bp, based on the site-exclusion model of
McGhee and von Hippel.30 Representative Scatchard
plots are shown in Figure S2 (Supporting Information),
while the data for DNA binding by 1 are summarized in
Table 1.
220-310 nm. For 1, excitation spectra were recorded at λem
)
450 nm, while for 2 the spectra were acquired at 560 nm. The
ratio of the fluorescence intensity of the bound versus free (I/
Io) was plotted against the excitation wavelength. Fluorescence
quenching studies were carried out with the minor-groove
binding agent distamycin (Sigma). Stock solutions of dista-
mycin were prepared in DMSO and diluted to 3 mM in buffer
([distamycin] determined by UV-vis, ꢀ305 ) 34 000 M-1 cm-1).31
Solutions of 1 (15 µM) and 2 (4 µM) were prebound to 10 bp
equiv of CT-DNA in 2.0 mL of buffer, and the distamycin
solution was added in 3 µM aliquots.
Resu lts
Hyd r olytic Sta bility a n d P r oton Affin ity of Ta m -
bja m in e E (1). To provide information on the aqueous
stability of 1, rate constants for the hydrolysis of the
enamine were determined by UV-vis spectroscopy. The
determinations were performed in the neutral to basic
pH range (7.0-10.5) at 25 °C and at pH 7.4 at 37.5 °C.
Figure 3 shows representative UV-vis data for the
conversion of 1 to the aldehyde 4 in aqueous buffered
media, pH 9.0, at 25 °C and ionic strength 0.1 M
(NaClO4). Monitoring the disappearance of the enamine
(392 nm) or the appearance of 4 at 362 nm provided first-
order rate constants in good agreement ((5%). All the
values of kobs (s-1) are summarized in Table S1 (Support-
The poor water solubility of prodigiosin 2 made the
DNA-binding studies difficult. Solutions (4 µM) could be
analyzed in aqueous media at pH 6.5, where the prodi-
giosin is predominately protonated.18 However, even at
4 µM solutions of 2 precipitated from solution at pH 7.4
and above. At pH 6.5, isobestic points were not observed
in DNA titration experiments. Figure 4 shows the
changes in the UV-vis spectra of 2 upon additions of 10
bp equiv of CT-DNA (A), polyd[G-C]2 (B), and polyd-
[A-T]2 (C). At this concentration, the AT polymer caused
(29) Albert, A.; Sergeant, E. P. The Determination of Ionization
Constants. Chapman & Hall: London, 1962.
(30) McGhee, J . D.; von Hippel, P. H. J . Mol. Biol. 1974, 86, 469.
(31) Chen, F.-M.; Sha, F. Biochemistry 1998, 37, 11143.

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6864 J . Org. Chem., Vol. 64, No. 18, 1999
Melvin et al.
Ta ble 1. Su m m a r y of DNA Bin d in g Da ta for
Ta m bja m in e E (1)a
c
DNA
[NaCl]b pH % H392
B shiftd K × 10-4 ne
CT-DNA
CT-DNA
CT-DNA
CT-DNA
poly d(GC)
poly d(GC)
poly d(AT)
poly d(AT)
10
10
6.5
7.4
7.4
8.5
6.5
7.4
6.5
7.4
15
28
20
25
24
32
20
26
3
4
5
2
6
7
9
5
8.6
4.0
0.9
1.0
7.3
3.1
13.2
7.4
3
100
10
10
2.6
3
10
10
10
a
All experiments were conducted in 50 mM buffer (MES at pH
b
6.5, MOPS at pH 7.4, and CHES at pH 8.5) at 25 °C. Concen-
trations of NaCl are in mM. c % hypochromicity (% H) refers to
the reduction in the extinction coefficient of the absorbance of 1
d
at 392 nm at saturation. Bathochromic (red) shift measured in
nm. e Binding constants were determined from the McGhee-von
Hippel equation, where K is the apparent binding constant (M-1
)
and n is the site size in DNA base pair (see Figure S2 in the
Supporting Information). The estimated error in the K values was
(5%.
a bathochromic shift of 20 nm (spectrum C), while shifts
of only 6 nm were observed for CT-DNA (spectrum A)
and polyd[G-C]2 (spectrum B). Near saturation (2/DNA
) 1:20 bp), red specks formed in the aqueous media,
suggesting that the prodigiosin 2/DNA complex precipi-
tated from solution. Attempts to circumvent this problem
by adding 20 vol % DMSO, dioxane, or triethylene glycol
failed to prevent precipitation. These factors prohibited
accurate determination of equilibrium binding constants.
In contrast to 1 and 2, the aldehyde precursor 4 showed
no changes in its absorption at 362 nm upon addition of
DNA (data not shown). As described in further detail
below, these results were consistent with the inability
of 4 to bind DNA effectively.
F lu or escen ce Sp ectr oscop y. In water, 1, 2, and 4
were found to exhibit weak emission spectra. Thus, we
sought to study their emission profiles in various media,
and Table 2 lists the relative emission intensities (com-
pared to that observed in ambiently oxygenated water)
for 1, 2, and 4 at a given concentration. Across the series,
the data shows that in going from H2O to MeOH to
CHCl3, the emission intensities increase, and this was
especially the case for the bipyrrole aldehyde 4. In MeOH,
increased concentrations of 2 quenched the fluorescence
spectra and caused a shift to longer wavelengths (Figure
5). The result in Figure 5 was attributed to aggregation
of the prodigiosin, as other cationic pigments, such as
ethidium32 and the cyanine dyes,33-35 are known to form
aggregates in solution that exhibit quenched fluorescence
compared to their monomeric counterparts.32-35 In CHCl3,
the effect of a proton donor (CH3COOH) on the emission
yields of 1, 2, and 4 was also examined. These experi-
ments were carried out to determine whether quenching
due to excited-state proton transfer36-38 plays a role in
the weak emission yields of these compounds in water.
However, in this case, additions of CH3COOH had
F igu r e 4. UV-vis spectra of prodigiosin 2 (4 µM) in the
absence (solid line) and presence (dotted line) of 10 bp equiv
of CT-DNA (A), polyd[G-C]2 (B), and polyd[A-T]2 (C) in 50 mM
MES buffer pH 6.5 containing 10 mM NaCl.
Ta ble 2. F lu or escen t Da ta for 1, 2, a n d 4 in Differ en t
Med ia a
medium
1 (15 µM)
2 (4 µM)
4 (5 µM)
H2O
MeOH
CHCl3
1.0
1.6
2.4
1.0
1.4
2.6
1.0
9.0
18.3
(32) Bresloff, J . L.; Crothers, D. M. Biochemistry 1981, 20, 3547.
(33) Khairutdinov, R. F.; Serpone, N. J . Phys. Chem. B 1997, 101,
2602.
(34) Smith, J . O.; Olson, D. A.; Armitage, B. A. J . Am. Chem. Soc.
1999, 121, 2686.
(35) Seifert, J . L.; Conner, R. E.; Kushon, S. A.; Wang, M.; Armitage,
B. A. J . Am. Chem. Soc. 1999, 121, 2987.
a
Values are given as relative emission intensities in the
indicated medium compared to that observed in ambiently oxygen-
ated water alone under the same experimental conditions. The
estimated error in the relative emission intensity values was (0.1.
virtually no effect on the emission spectra of 1, 2, and 4
(data not shown).
We then utilized fluorescence spectroscopy to study
DNA binding by 1, 2, and 4. Addition of excess DNA (10
(36) Liu, W.; Welch, T. W.; Thorp, H. H. Inorg. Chem. 1992, 31, 4044.
(37) Goll, J . G.; Liu, W.; Thorp, H. H. J . Am. Chem. Soc. 1993, 115,
11048.
(38) Turro, C.; Bossmann, S. H.; J enkens, Y.; Barton, J . K.; Turro,
N. J . J . Am. Chem. Soc. 1995, 117, 9026.